express

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m.kh.rashidi
m.kh.rashidi's picture
express

Hi
I have a problem in gene expression.
It is about 9 months that I am unsuccessful in expression.
my target gene is Eukaryotic and 405bp.
And I optimized all of conditions that are necessary for expression Eukaryotic gene in bacterial cell like codons usage, GC content , mRNA folding and etc.
Gene expression vectors that I used for cloning are
PET 28, 32, 22 and PGEX 4T-1 vector.
the cloning Confirmed by PCR and digest .
Expression conditions:
OD: different condition(0.4-1) 620nm
IPTG: different condition (0.3-1)Mmol/lit
incubation Temperatures: different condition(22-37) C
Incubation times: 2-24h
competent cell for transformation BL21?DE3.
It should be noted that when the empty vector(PET32) was transformed in to competent cell with above condition was expressed.
However, the cloned gene is not expressed.
Thank you.

Jason King
Jason King's picture
 What does the 405bp code for

 What does the 405bp code for? Is it a membrane protein? 

If it can be bought commercially, is it very expensive - if so then there is likely to be a well known problem in its expression. The solution might be to move to a eukaryotic expression system.

m.kh.rashidi
m.kh.rashidi's picture
Hi

Hi
This gene encodes a transmembrane protein
A marker for colorectal cancer and its molecular weight 14 KD and pH I 9.5.
The native form is a glycoprotein with a molecular weight of 46KD
Two subunits are connected by disulfide bonds.